According to the prion hypothesis, an alternative conformation of a normal, host-encoded protein, known as a prion, can function as an epigenetic determinant of transmissible phenotypic states in vivo. Once considered atypical, this process has been linked to an expanding range of previously enigmatic biological phenomena, such as the etiology of the transmissible spongiform encephalopathies (TSEs) and the non-Mendelian inheritance of a group of metastable traits in fungi, suggesting it is a widespread process through which organisms can access and perpetuate parallel phenotypic states. While the protein-only basis of these phenotypes is well documented, we have surprisingly limited insight into how transitions between them occur, despite their significance for normal cellular physiology. Our long-term goal is to reveal the pathways through which prion transitions are evoked in vivo. To gain this insight, we will exploit the manipulability of the Sup35/[PSI+] prion of S. cerevisiae. Prion propagation in this system, and also likely in mammals, occurs through a multi-step pathway of protein biogenesis that is influenced by both the inherent properties of the prion and by aspects of the cellular environment. [PSI+] propagation is normally efficient, but many conditions, with parallels in mammals, induce phenotypic transitions. The overall objective of this proposal is to combine empirical and computational studies to determine which step(s) of the in vivo prion propagation pathway are altered by these manipulations and the cellular pathways through which these changes evoke the switch. To date, most analyses have considered these effects from the perspective of the prion alone, but our proposed studies consider their action in the context of the entire system. While conditions that induce transitions between prion-associated phenotypes introduce different conformational or sequence variants of the prion into cells, the role of competitive forces in prion transitions has never been explored. We hypothesize that the crucial steps in the prion propagation pathway should be considered as enzyme-limited processes that are subject to competitive forces, which can effectively evoke phenotypic transitions. To directly test this hypothesis, we will determine: 1) the pathways by which dominant-negative mutants cure prions, 2) the pathways by which prion variants establish dominance, and 3) the molecular basis of the requirement for an existing prion in the de novo appearance of a second prion. Through this unique perspective, we will begin to reveal the cellular pathways underlying transitions between prion-associated phenotypes, a crucial yet poorly understood aspect of prion biology. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the appearance of alternative, self-replicating protein conformations are associated with a wide array of familial, sporadic and transmissible neurodegenerative diseases in man. Our current inability to explain, predict or exploit transitions between normal and disease states is a critical barrier to progress in the development of treatments for these disorders. Our proposed studies are relevant to the mission of the NIH because the knowledge gained here will provide insight into the forces constraining switches between these states and the robustness of the system to external perturbation.